Expression and application of high-glucose-resistant and alkali-resistant [beta]-glucosidase

A technology of glucosidase and high sugar resistance, applied in the field of β-glucosidase, which can solve the problem of no transglycoside activity

Inactive Publication Date: 2015-11-11
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This β-glucosidase is very glucose t...

Method used

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  • Expression and application of high-glucose-resistant and alkali-resistant [beta]-glucosidase
  • Expression and application of high-glucose-resistant and alkali-resistant [beta]-glucosidase
  • Expression and application of high-glucose-resistant and alkali-resistant [beta]-glucosidase

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Experimental program
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Effect test

Embodiment 1

[0024] Obtaining the amino acid sequence of β-glucosidase glA2

[0025] The genomic DNA of anaerobic bacillus yellow fever anaerobic bacillus yunnanensis subspecies PGDY12 was extracted according to the instructions of the Ezup Column Bacterial Genomic DNA Extraction Kit (Shanghai Sangong Biotechnology Co., Ltd.). According to NCBI, anaerobic bacillus yellow fever subspecies Yunnan E13 T The upstream and downstream primers of PCR were designed for the gene sequence of β-glucosidase, and their sequences were respectively: 5'-ATGCTTCAGTTTCCGAAA-3' and 5'-TTTAACTCCATGATTCATG-3'. Using the extracted genome as a template, the product obtained by PCR amplification was sent to Shanghai Sangong Biotechnology Co., Ltd. for gene sequencing, and the amino acid sequence and wild-type gene sequence.

Embodiment 2

[0027] Gene synthesis of β-glucosidase glA2

[0028] Since the protein expressed by the wild-type gene sequence of the β-glucosidase in Escherichia coli is an inactive precipitated inclusion body, the wild-type gene sequence was optimized. The principle of optimization is mainly to redesign the gene sequence of glA2 according to the codon preference of Escherichia coli, and at the same time optimize the secondary structure of the N-terminal mRNA by 6 nucleotides, and finally obtain the gene shown in SEQ ID NO: 1 sequence. The similarity between the gene sequence and the wild-type gene sequence is only 75.2%, which is similar to that of Bacillus ylavotherii subspecies Yunnan subspecies E13 T The gene sequence similarity of β-glucosidase among them was 74.9%. Add NdeI and XhoI restriction enzyme cutting sites at both ends of the gene. The text sequence of the gene was sent to a biological company (Shanghai Sangon Biotechnology Co., Ltd.) for synthesis. The synthetic glA2 gen...

Embodiment 3

[0030] Construction of expression vector pET22b-glA2 of β-glucosidase glA2

[0031] The cloning vector pUC57 containing the glA2 gene and the expression plasmid pET22b were digested with NdeI and XhoI respectively, and after electrophoresis separation of the digested products, the glA2 gene fragment and the expression plasmid pET22b were recovered. Add T4 DNA ligase 16. °C for overnight connection. The ligated product was transformed into Escherichia coli BL21 (DE3) competent cells, positive clones were screened out, and the recombinant expression plasmid pET22b-glA2 was obtained.

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Abstract

The invention discloses [beta]-glucosidase glA2, of which the gene sequence is represented as the Seq ID No.1. An amino acid sequence encoded by the gene sequence is represented as the Seq ID No.2. Comapred with the [beta]-glucosidase in the prior art, the [beta]-glucosidase glA2 has following advantages: (1) the [beta]-glucosidase glA2 can be improved in enzyme activity significantly with glucose, even that the concentration of the glucose is lower than 1.75 mol/L, and when the concentration of the glucose is 0.4 mol/L, the enzyme activity can reach up to 136.8 U/mg, which is 2.2 times as the enzyme activity without glucose; and (2) the [beta]-glucosidase glA2 is excellent in alkali stability, wherein the enzyme activity can be maintained by 92%, 99% and 97% even that the enzyme is incubated under the pH respectively being 8.0, 9.0 and 10.0 for 24 h. The [beta]-glucosidase glA2 can be applied in a catalytic environment contains high-concentration glucose or is alkaline.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a beta-glucosidase, which can significantly increase the enzyme activity by glucose and maintain excellent stability under alkaline conditions. Background technique [0002] β-glucosidase (β-glucosidase, EC3.2.1.21), also known as β-D-glucoside glucohydrolase, is an exohydrolase. It can hydrolyze the non-reducing β-D-glucosidic bond bound to the terminal, and is widely used in the release of food flavor substances, cellulose degradation and daily chemical industry. In these applications, glucose is often one of the products of hydrolysis, but glucose is also an inhibitor of most β-glucosidases. Therefore, excavating β-glucosidases with high glucose tolerance has always been a research hotspot. [0003] In the current literature reports, there are more than forty microorganism-derived β-glucosidases with different degrees of glucose tolerance. There are only 3 β-glucosidases whose ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12R1/19
Inventor 彭惠易路张销寒高毅
Owner ANHUI UNIVERSITY
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